The present invention relates generally to peptide fragments and synthetic analogs of thrombospondin (also known as thrombin sensitive protein or TSP) with thrombospondin-like activity. The peptides mimic or inhibit the biological activity of TSP. These peptides may be used in biological and pharmaceutical applications such as: (a) inhibiting the invasive and metastatic activity of melanoma cells, (b) promoting and inhibiting cellular attachment to tissue culture flasks, (c) promoting wound healing, angiogenesis, and implant acceptance, (d) agents for anti-platelet aggregation, (e) agents for antimalarial activity, and (f) diagnostic reagents in different therapeutic applications, as well as other related areas.
Thrombospondin (TSP) is secreted by platelets in response to physiological activators such as thrombin and collagen (Lawler, Blood, 67:112-123 (1986)). Other cells also synthesize TSP including fibroblasts (E. A. Jaffe et al., Proc. Natl. Acad. Sci., 80:999-1002 (1983)), smooth muscle cells (Raugi, G. J. et al., J. Cell Biol. 95:351-354 (1982)), and endothelial cells (J. McPhearson et al., J. Biol. Chem., 256:11330-11336). TSP has been found in certain tumor tissues, such as melanoma cells (J. Varani et al., Clin. Expl. Metastais, 7:265-76 (1989)), squamous lung carcinoma (B. L. Riser et al., Exp. Cell Res., 174:319-329 (1988)) and breast carcinoma (D. A. Pratt et al., Eur. J. Cancer Clin. Oncol. 25:343-350 (1989)). In addition, certain tumor cells in culture, such as, fibrosarcoma, rhabdomyosarcoma, glioblastoma, Wilm""s tumor, neuroblastoma, teratocarcinoma, choriocarcinoma, melanoma, and lung carcinoma have been shown to synthesize TSP (D. F. Mosher, Annu. Rev. Med., 41:85-97 (1990)).
TSP has been shown to play a role in many diverse and clinically important processes, such as: cell migration, wound healing, nerve regeneration, and tumor cell metastasis. TSP has been purified by a number or procedures including exclusion chromatography (Lawler et al., J. Cell Biol., 103:1635-48 (1986)). The complete amino acid sequence of TSP has been deduced from DNA clones prepared by various groups including Lawler et al., J. Cell Biol., 103:1635-48 (1986); Kobayashi et al., Biochemistry, 25:8418-25 (1986); Dixit et al., Proc. Ntl. Acad. Sci., 83:5449-53 (1986); and Hennessy et al., J. Cell Biol., 108:729-36 (1989). The structure of TSP is conserved among various animal species as indicated by the fact that the antiobody against the human protein cross-reacts with TSP from mouse, rat, pig, cow, sheep, dog, and turkey (H. I. Switalska et al., J. Lab Clin. Med; 106:690-700). It is now known that TSP, originally characterized from platelet released proteins, is only one member of a family of structurally related proteins encoded by different genes which include at least four new members designated TSP-2, TSP-3, TSP-4, and TSP-5/COMP (cartilage oligomeric matrix protein). Adams and Lawler, Current Biology, 3: 188-190 (1993).
TSP-1 is composed of three identical disulfide-linked chains each consisting of 1,152 amino acids (MW 145,000), and each polypeptide chain is composed primarily of domains consisting of repeating homologous amino acid sequences. Adams and Lawler, Current Biology, 3: 188-190 (1993). These domains are a) NH2-terminal globular domain; b) a procollagen homology domain; c) the type 1 or properdin repeat domain, consisting of three repeating sequences homologous to sequences found in properdin; d) the type 2 repeat domain, consisting of three repeating sequences homologous to those in epidermal growth factor; e) the type 3 repeat domain, consisting of seven repeating Ca2+-binding sequences, and f) a COOH-terminal globular domain. These distinct domains interact with different cell surface receptors and mediate a variety of cellular processes including cell attachment, migration, proliferation and differentiation. For example, the Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) sequence within the type 1 repeats binds the Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1) receptor, recently identified and isolated from tumor and endothelial cells (Tuszynski et al., J. Cell Biol., 116:209-217 (1992) and Prater et al., J. Cell Biol., 112: 1031-1040 (1991) and is part of the central stalk or protease resistant region of TSP-1.
The present invention provides thrombospondin fragments and analogs that mimic or inhibit the biological activity of intact thrombospondin and are, thus, useful in a variety of biological, prophylactic or therapeutic areas. These peptides are capable of modifying and inhibiting tumor cell metastasis, cell adhesion and platelet aggregation in mammals in vivo. The peptides are also useful in wound healing, for antimalarial activity, atherosclerosis, thrombotic, and thrombolytic conditions, angiogenesis, and as cell attachment promoters, complement modulators, and diagnostic reagents and in other related areas.
Analogs based on the type I repeat of thrombospondin described by Lawler et al., Seminars in Thrombosis and Hemostasis, 13:245-254 (1987), Robson et al., Nature, 335:79-82 (1988), and Groundis et al., Nature, 335:82-85 (1988) have been shown to have thrombospondin-like activity. Specifically, analogs based around and including at least a portion of the sequence motif Trp-Ser-Pro-Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 2) have been shown to have thrombospondin-like activity.
This invention is directed to polypeptide compounds of formula (I):
Z1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Z2xe2x80x83xe2x80x83(SEQ ID NO: 3)
wherein:
Xaa2 is a neutral/non-polar/large/cyclic amino acid residue;
Xaa3 is a neutral/polar/small or neutral/polar/large/non-cyclic or acidic amino acid residue;
Xaa4 is a neutral/nonpolar/large/cyclic or neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;
Xaa5 is a neutral/polar/small amino acid residue
Xaa6 is a neutral/polar/small or neutral/polar/large/non-cyclic amino acid residue;
Xaa7 is a neutral/nonpolar/large/non-cyclic or neutral/polar/large/non -cyclic amino acid residue;
Xaa8 is a neutral/polar/large/non-cyclic or neutral/polar/small amino acid residue;
Xaa9 is a neutral/polar/small amino acid residue;
Xaa10 is a neutral/polar/small amino acid residue;
Z1 is hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;
Z2 is hydroxyl, carboxyl, non-amino acids such as agmatine, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof.
Preferably, the polypeptide compounds of this invention have formula (II):
R1-Cys-Xaa11-Xaa12-Xaa13-Cys-R2xe2x80x83xe2x80x83(SEQ ID NO: 4)
wherein:
R1 is a protected or unprotected terminal amino group, including hydrogen, amino, acetyl or at least one amino acid residue or the desamino form thereof;
Xaa11, Xaa12, and Xaa13 are the same or different neutral/non-polar/large/non-cyclic or neutral/polar/large/non-cyclic or neutral/polar/small or basic/non-cyclic amino acid residues, preferably selected from the group consisting of valine, threonine, serine, and arginine;
R2 is a protected or unprotected terminal carboxyl group including hydroxyl, carboxyl, or at least one amino acid residue, including carboxyamide or alkylamide forms thereof, preferably selected from the group consisting of lysine, glycine, and arginine;
wherein the structure of the polypeptide is optionally cyclized through a bond between the cysteines, such as a disulfide bond, or a bond between R1 and R2.
This invention also includes polypeptides having the retroinverso form of L-amino acid polypeptides of formulae (I) and (II), i.e, polypeptides comprising D-amino acids in reverse order. In particular, retroinverso peptides of formula Cys-Ser-Val-Thr-Cys-Gly (SEQ ID NO: 1), i.e., d-Gly-Cys-Thr-Val-Ser-Cys (SEQ ID NO: 5) are preferred. Preferably, the cysteine residues are modified by a sulfhydral blocking group, such as xe2x80x94CH2xe2x80x94NHxe2x80x94COCH3, that is adhesive toward melanoma cells. Alternatively, the cysteine residues may be conservatively substituted with another amino acid, e.g., methionine.
Also provided in accordance with aspects of the invention are pharmaceutical compositions, which contain the above-recited polypeptide compounds together with a pharmaceutically acceptable liquid, gel or, solid carrier. Administration of therapeutically effective doses of these compositions can provide effective enhancement or inhibition of thrombospondin-like activity to animals, particularly vertebrates such as mammalian and avian hosts.